Developmental mechanisms of differentiation can be divided into two types: the generation of distinct cell types from a single multipotential progenitor cell, and the sequential stages in the process of generating a specific cell type. B cell development, as do development of other cells of the hematopoietic system, offers a unique opportunity to study both mechanisms. B cell development can be divided into several stages: commitment to B cell development, progenitor B cells (pro-B), pre-B cells and mature B cells. The transcription factors involved in early B cell development are relatively well characterized, but less is known about later stages of this development. I propose to study the mechanisms involved in pre-B cell stage to B cell transition. I will use a knockout mouse described recently at Dr. Singh's lab. These mice have had IRF4,8 deleted, causing a pronounced pre-B cell stage arrest, which offers a unique opportunity to study this cell stage. I will first characterize the molecular phenotype of IRF4,8-/- pre-B cells, especially what is preventing light chain rearrangement, a major event that should occur at this stage. Then I can use these mice to identify genes involved in pre-B cell stage to B cell transition, and to analyze the temporal order of events in this transition. My work until now had been to study how one factor - DNA methylation - affects gene transcription. In this work I will study how different transcription factors work together to create the correct gene transcription pattern. This combined knowledge - of how single elements work, and how different elements work together, will enable me to understand complex biological systems.

I propose to study the serotonergic modulation of C. elegans locomotory behavior using optical activity recording and optical stimulation of specific neurons together with pharmacological, molecular and genetic tools. A genetically-encoded voltage sensitive probe will be expressed under specific promoters in sensory and interneurons and used to record neuronal activity. This will enable the identification and characterization a postulated locomotory CPG and permit characterization of network activity under various conditions. A transgenic photostimulation system will be also expressed in specific sensory and interneurons to stimulate them in a noninvasive manner. The effect on the nematode behavior and neuronal activity will elucidate the locomotory network and its functional connectivity. Pharmacological and genetic manipulations will be used to dissect different aspects of the 5-HT modulation of behavior. Lastly, a naturally occurring 5-HT hypersensitivity allele will be cloned. Apart from my 3-month long Grass Fellowship, I had never studied C. elegans, applied molecular techniques nor used activity imaging. This is a good opportunity to fill the molecular and genetic gap in my scientific background. Also, I will be able to take these optical tools for recording and stimulating neuronal activity in C. elegans and use them when I start my own laboratory and share them with colleagues. C. elegans is a very promising neuroethological model. In several years it will probably be the first animal in which all neurons will be recorded at once during natural behavior and behavioral plasticity.

Humans, more than any other primate, are first and foremost social creatures. Yet we still have little understanding of the neural basis of social cognition and behavior. The research proposed here belongs to an emerging area of interdisciplinary research, social neuroscience. My aim is to develop new techniques for studying social behavior, based on brain imaging (fMRI). Specifically, I wish to search for neural correlates of processes such as social imitation, social skills and the implicit and explicit effects of contextual factors on our social behavior. In my PhD I studied the organization and function of high-order visual regions, under highly controlled and exceedingly simplified settings, while treating each subject in isolation. However, our most basic human traits - our thoughts, goals, intentions and feelings - are influenced by our social and cultural environment. In my last PhD project I therefore developed methods to study brain activity with more natural stimuli and viewing conditions, and showed their validity by replicating results from my previous work. I propose to use this "natural viewing" paradigm to study the pathways by which social processes influence and are influenced by brain function. The development of a new field, especially one that spans disciplinary boundaries, takes time. My work will advance collaboration between three leading scientists in social psychology and neuroscience. I believe that the proposed project, though ambitious, is feasible with this mentoring team, and will result in a better understanding of how the brain performs its social functions.

A longstanding aim in neuroscience is to find neuronal substrates of behaviors. Here, we (i) explore a novel strategy to identify and functionally characterize the neuronal circuits involved in specific behaviors in genetically modified mice and (ii) apply this method to investigate the role of the Amygdala central nucleus (CeA) in innate vs. conditioned fear. We propose to use genes specific for neuronal populations that show behavior induced activity, to express genetically encoded trans-synaptic tracers to map the neuronal circuitry the neurons are part of. Similarly, expressing an inducible genetically encoded neuronal silencer under control of the same genes will allow silencing of this circuit in behavioral paradigms for innate and learned fear. Thus, we hope to functionally dissect the role of the CeA in these two forms of fear to better understand the neuronal basis of anxiety disorders in human. The proposed research will extend my training to behavioral neuroscience and thus complements my previous experience in functional and developmental neuroscience. Both topic and approach, will be central to my future research outlined previously. Future directions, using similar strategies that combine molecular genetics, genomics and behavioral neuroscience, are (i) the identification and functional characterization of genes – associated with altered brain function in evolution or disease - and (ii) to exploit across species gene expression data (human, chimp, mouse) to identify homologous neuronal structures in mice (even if they cannot be defined anatomically) and then address human specific traits in the mouse as model system.

Meiosis I is fundamentally different from meiosis II and mitosis in that homologous chromosomes, not sister chromatids, are segregated to opposite poles. By using fission yeast as a model system, I want to study the mechanisms of this unique chromosome segregation. I will carry out two genetic screens to find novel factors that are involved in setting up the specialized chromosome segregation of meiosis I. Specifically, I want to know what allows sister kinetochores to be oriented towards the same, and not opposite spindle poles during meiosis I, and I want to identify genes required for sensing and translating chromosome attachment errors typical for meiosis I. The evolutionary conserved Aurora kinase is a candidate for being involved in the latter process, and I will therefore also examine the function of this protein kinase in meiosis.

Neuronal variability, which is necessary for the proper assembly of neuronal connections and the correct projection to different specific targets in the brain, is, at least in part, a consequence of differential regulatory gene expression and downstream control of axon guidance molecules during development. The goal of my research is understand the mechanisms underlying neural identity and how they lead to different projection phenotypes. I plan to approach the problem using a different system and a new perspective compared to those used by other labs. While work from other groups has outlined programs of cell type specification and directives for axon growth to peripheral targets using the spinal cord motoneurons or the visual projections to the cortex as a model, I will use the mouse visual system to focus on the identity among retinal cells resulting in specific axonal projection patterns with respect to laterality at the midline. I will then extend these studies to other binary decision points through the developing nervous system, specially the patterning of bilateral organization of other sensory systems in the CNS. This work will primarily shed light on a long-standing unsolved question: how are retinal axons specified to diverge to both sides of the brain? Deciphering this question should also provide information on disruption of visual routes by genetic perturbations such as albinism. Moreover, since Zic2, which controls the uncrossed retinal projection at the chiasm, is also responsible for severe malformations as holoprosencephaly and spina bifida in humans, my future work on Zic2 related genes in retina could help in the investigation of these anomalies

The goal of my proposed project is to understand the role of molecular chaperones in the maintenance of the prion state. I will use Saccharomyces cerevisiae as a model system and focus on the yeast prion [RNQ+] maintenance modulated by Hsp70 Ssa, using various analysis methods of genetics, cell biology, molecular biology, biochemistry, and physical chemistry. Firstly, I will isolate SSA mutants defective in maintenance of the prion state in yeast, and then characterize these effects using cell biological techniques. Next, to understand the mechanism at a molecular level, I will purify these mutants and characterize them in vitro assay systems using biochemical techniques. This project will provide us both fundamental understanding of the prion propagation mechanism in a cell and thus potentially have important implications for medicine and biotechnology. I have studied physical properties of protein in vitro using physicochemical and physical techniques, whereas in my proposed project, I will study protein functions in the cell using cell biological methods. Thus, there are significant differences between my previous research and my proposed project in both research direction and method. I am eager to acquire expertise in the cell biological and genetics field, in addition to the physicochemical and biophysical skills I already have. I think that a cell biological and molecular biological approach combined with in vitro biochemical and physicochemical techniques is important for unraveling the complex functions of living organisms. The HFSP fellowship will provide me a great chance to obtain training in a new area of research and to perform combined analysis approaches.

Malaria afflicts between 300 and 500 million people annually. The Plasmodium parasites responsible for malaria have demonstrated increasing drug resistance, suggesting that in time current treatments will be rendered obsolete. Malaria is almost exclusively confined to developing countries, and has been neglected by most major pharmaceutical companies. This project is aimed at creating new inhibitors for the malarial protease plasmepsin II and evaluating their effect on the pathology of Plasmodium parasites. The inhibitors will be devised using computer-aided de novo design, and made by standard synthetic methods. Evaluation will begin with in vitro determination of their ability to inhibit plasmepsin II. Biophysical characterization of the inhibitor-enzyme interactions will be carried out using crystallography and calorimetry. Finally, the effect of such inhibitors on parasite pathology will be examined. My previous research concerned the creation and study of self-organizing container molecules, and was driven by curiosity about the physical behavior of molecules confined within nanoscopic spaces. The present research is interdisciplinary, and through it I hope to gain experience with a variety of biochemical and biological techniques: computer-aided protein modeling, protein activity assays, protein crystallography, isothermal titration calorimetry, and small animal disease models. In the next stage of my career, I hope to use this experience to carry out research at the interface of chemistry and biology.

Our ability to store new information and memories is believed to require a property of certain neurons called synaptic plasticity--the ability to specifically and dynamically alter pre-existing connections between neurons. While it is known that electrical and chemical signals underlie synaptic plasticity the precise molecular components regulating this process are largely uncharacterized. I propose to investigate how synaptic plasticity is affected by the organization of different molecules at synapses. My post-doctoral research will focus on Ras and Rap1/2, proteins which appear to have the ability to change the physical structure of neuronal connections. This research will extend my previous experience in characterizing regulators of neuronal architecture in vitro to a study investigating the relationship between synaptic plasticity and molecules which alter synaptic structure in vivo. This post-doctoral investigation will allow me to synthesize several different scopes of research including biochemical and molecular aspects I focussed on in my PhD, and will develop my expertise in live imaging, production of knockout mice, and ultimately in behavioural analyses. These studies will not only deepen our understanding of synaptic plasticity and memory formation, but could ultimately provide the insights needed to counter injuries and combat diseases that impair memory. The knowledge I anticipate gaining from these studies under the direction of Dr. Morgan Sheng at MIT will better prepare me to direct my own academic research in which I plan to investigate the complexities involved in the function and integration of cellular signalling systems within an entire organism.

1) Proposed project: This project is the first approach to genetically dissect the mechanisms of metastasis in Drosophila. To identify the genes regulating metastasis, I will perform a large-scale genetic screen using a Drosophila cancer model. I will utilize a Drosophila model of benign tumor established in Dr. Tian Xu’s laboratory, and conduct a genetic screen to identify mutants acquiring metastatic features in these tumors. I will genetically isolate responsible genes for the ‘metastasis promoting’ mutations, and dissect the signaling pathways of metastasis both genetically and molecularly. I believe that it would bring a breakthrough in this research field. 2) How it differs from my previous research experience: So far I have studied the mechanisms of cell death in mammals and Drosophila. Taking advantage of powerful genetics of Drosophila, I propose a novel approach dissecting the mechanisms of metastasis through genetics. Metastasis is achieved by several stepwise progressions of tumor cells including cell growth, invasion and cell migration. This line of biological phenomenon can be accomplished independently of cell death machinery, indicating this research field is completely different from all my previous experiences. 3) How it will serve my professional growth: Through this project, I will experience highly complicated genetics and strategies against complex biological phenomena in whole animals. In addition, the genetic approach to understand the mechanisms of metastasis will provide me an experience to study various biological features of cells and their signaling pathways that I have never studied. These aspects will strongly support my professional growth.

1) To become functionally active, newly synthesized protein chains must fold to unique three-dimensional structure, but regulated unfolding also is critical during the life cycle of many proteins. Some unfolding processes show surprising similarities. For instance, many proteins in degradation and membrane translocation are targeted for unfolding by terminal signal sequences and translational movement of the substrate protein often occurs with unfolding. Taking together the previous results, the susceptibility of proteins to unfolding by protease and translocase depends on the proteins' stability against global spontaneous unfolding and, more importantly, on their local structure adjacent to the targeting signal. I assume that the unfoldases trap local unfolding fluctuation in the part of the substrate structure adjacent to the targeting signal effectively pulling at the polypeptide chain. To test this hypothesis, I will measure both the local unfolding fluctuations adjacent to the targeting signal in substrate proteins by using NMR spectroscopy as well as the physical stability of substrate proteins against pulling in atomic force microscopy experiments. 2) & 3) I have worked on the biophysical side of molecular chaperone which assists protein folding and my host laboratory has worked on biochemical and molecular biological as well as biophysical aspects of in vivo protein unfolding. Although protein unfolding is an opposite phenomenon to protein folding, my biophysical background will allow me to make unique contribution to biologically important in vivo protein unfolding and I will broaden my research experience to include in vivo protein unfolding.

In the proposed project, I will investigate fundamental questions concerning the link between behavior and neuronal activity in the brain. In order to address these questions, I will develop novel techniques for combining of electrical and optical recording from rodent cerebellum in vivo. The activity of the cerebellar cortical network will be visualized using voltage-sensitive dyes and a high-speed cooled CCD camera. At the same time, cellular and synaptic activity will be investigated using patch-clamp recordings from single neurons combined with two-photon microscopy. Changes in the activity of cerebellar cortex induced by sensory stimulation will be captured by these techniques. I will also try to induce long-term depression at the parallel fiber and Purkinje cell synapse by applying a paradigm of classical conditioning in order to test whether simple motor learning is associated with plastic changes at the cerebellar synapses. Until now I have been working on synaptic transmission in the brain slice preparation using patch-clamp methods. The framework of the proposed project is consistent with that of my previous work in the sense that it investigates synaptic modulation using electrophysiology. However, the project also offers me a transition from the slice preparation to the intact brain in vivo, with imaging techniques paired with electrophysiology. These technical developments will allow me to relate the synaptic dynamics that I have been studying to the function of neural networks and ultimately to behavioral events. This project will therefore enable me to expand the scope of my research and gain a wider range of experience in the field of neuroscience.

The ultimate goal of this research proposal is to dissect the molecular nature and the post-transcriptional regulation of two proteins, ABI5 and its interacting partner AFP, that are critically involved in ABA signaling pathway. This work is of significance because these proteins have been shown to interact in vivo at the sites of nuclear bodies where ubiquitin-mediated proteolysis can possibly occur. An understanding of the role of regulated proteolysis in ABA signaling will shed light on the importance of similar processes in plant developmental pathways. In addition, nuclear body speckle formation has been shown to be associated with the modification of proteins by SUMO. My experiments will demonstrate if AFP is a substrate for SUMO and if this modification has functional significance. Thus, this research proposal will not only help to define an important regulatory step in the ABA response pathway, but also offer wide vistas into novel mechanisms for maintaining homeostasis during plant development. As a doctoral student, I have studied the role of histone deacetylase in rice development and investigated the improvement of transgene expression in rice. Hence, my research experiences have been restricted to biotechnology based on plant biology and agricultural science. The opportunity to study the roles of regulated proteolysis in ABA signaling in Dr Chua’s lab will expand my scientific horizon with respect to learning new concepts in cell and molecular biology and acquiring additional technical skills. These experiences will greatly enhance my development as an independent investigator upon the completion of my post-doctoral training.

Cell lineage specific transcriptional programmes are set early in development and maintained, via epigenetic mechanisms, throughout the lifetime of an organism. The failure of maintaining &#8216 cell memory&#8217 may result in cell identity deviance, deregulation of cell proliferation, altered developmental programmes and cancer. Modulation of chromatin structure appears to be a key pathway controlling transcription memory. The balanced action of chromatin regulators of the PcG and trxG groups controls the expression of homeotic as well as of most developmentally regulated genes. There is an increasing evidence that PcG/trxG genes might be involved in control of cell proliferation and cell cycle progression, including stem cell renewal and tumor progression. By combining molecular (chromatin immunoprecipitation, RNAi/ribozymes) and cytological (BrdU-incorporation, histone modifications immunodetection) approaches, the aim of this project is to get more insight into the role of PcG/trxG proteins in cell totipotency and epigenetic levels of genome reprogramming. The proposed research would significantly broaden my experimental skills, and challenge entirely new aspects of genome function, namely how epigenome reprogramming may influence cell identity definition and redefinition.

Synapses in a nervous system form specific connections between subsets of neurons. Once an axon has been guided to its target region, molecular cues and neuronal activity control the formation of synaptic contacts with only a part of the possible target cells. The goals of the proposed project are (i) to identify the cell surface molecules involved in selective synaptic targeting and (ii) to characterize their molecular activities using the fruit fly Drosophila melanogaster as a model system. The fly olfactory system represents a genetically tractable system for rapid identification of novel cell surface molecules involved in target specificity. I propose to establish a genetic screen based on fluorescent synaptic markers expressed in two olfactory receptor neurons. In a subsequent line of experiments I will apply the tandem-affinity purification-tag methodology to purify interactors of candidate cell surface proteins identified in the screen. Previously, I focused on the role of cell surface molecules in the adult nervous system using mouse genetics, biochemical assays, and electrophysiology. The proposed project aims at understanding a developmental process using completely different techniques in the fly nervous system. Thus, this fellowship provides the opportunity of training in complementary techniques, which will prepare me excellently for my long term goal to establish my own laboratory. These experiments will give novel insights into mechanisms involved in target selectivity of synapses and furthermore, given the fundamental conservation between Drosophila and higher mammals, provide candidates for evaluation in mammalian nervous system development.